Inactivation of Retinoblastoma (Rb) tumor suppressor pathway is an early obligatory event in human cancer. One of the key targets of Rb pathway is the family of E2F transcription factors that regulates cell proliferation and apoptosis. Overlapping and redundant functions of mammalian members of E2F and pRB families often complicate identification of their in vivo function. Model organisms such as Drosophila provide attractive alternatives to complement studies in mammalian systems due to high conservation of Rb pathway and amenability of flies to genetic analysis. Interestingly, Drosophila E2F gene dE2f1 contains an intronic microRNA cluster mir11~998. Such arrangement sets a stage for possible functional interaction between the miRNA and its host gene. Surprisingly, even though more than half of vertebrate miRNAs are intronic this issue remains largely unappreciated and therefore this class of miRNAs is rarely investigated in the context of the gene they reside in. We have recently discovered that the mir11~998 cluster is intimately involved in modulation of E2F dependent apoptosis. miR-11 directly regulates expression of apoptotic E2F targets to limit irradiation induced apoptosis. Unlike miR-11, miR-998 operates through a different mechanism. In preliminary experiments, we have found that miR-998 regulates E2F dependent apoptosis by modulating the prosurvival EGFR signaling. We will build on these initial findings to establish the functional significance of regulation of the mir11~998 cluster embedded into the dE2f1 gene. In Aim 1, we will validate dCbl as a direct target of miR-998 to mediate its effect on EGFR signaling in Drosophila. In Aim 2, we will extend our work to mammalian cells and will evaluate the link between miR-29, a mammalian miR-998 homolog, and EGFR signaling. In Aim3, we will investigate the co-regulation of the mir11~998 cluster and its host gene dE2f1. We will determine the functional significance of interdependence between miR-11 and miR-998 within the cluster using novel knock-in alleles. Overall, this study will address largely unappreciated relationship between embedded miRNAs and their host gene and will evaluate the significance of such interaction in the context of Rb tumor suppressor pathway. Notably, miRNAs are known to influence various biological processes and play a crucial role in human malignancies such as cancer. Therefore the knowledge generated upon successful completion of this project will likely stimulate further studies linking miRNA regulation to E2F and may help in identifying novel therapeutic targets.